A hemocytometer is a specialized glass slide used for manually counting cells in a liquid sample. This tool determines the total cell count and viability of various cell types, making it important across many scientific fields. Widely used in cell culture, microbiology, and clinical diagnostics, hemocytometers provide a direct visual assessment of cells. Accurate cell counting ensures experimental reproducibility in research and supports patient care.
Hemocytometer Components and Design
A hemocytometer is a thick glass microscope slide with an etched grid on its surface. The Improved Neubauer chamber, a common type, features an H-shaped groove defining two counting chambers. Each chamber has a precise depth of 0.1 mm, crucial for cell concentration calculations.
The counting grid within each chamber contains nine large squares, each 1 mm by 1 mm. These large squares are subdivided into smaller squares for easier counting. For instance, the central large square is often divided into 25 medium squares, which are then divided into 16 smaller squares. This grid system, combined with the chamber depth, creates a known volume over each square, enabling accurate cell density calculations.
Preparing Your Cell Sample
Sample preparation is essential before loading the hemocytometer. Cells must be in a single-cell suspension, free of clumps, for accurate and even distribution. Thorough mixing of the suspension immediately before sampling prevents cells from settling.
Dilution is often necessary to achieve a countable cell density, typically 2.5 x 10^5 to 2.5 x 10^6 cells/mL for an Improved Neubauer chamber. High concentrations make counting difficult, while low concentrations increase random errors. Record any dilution factor accurately, as this will be used in the final cell concentration calculation.
For assessing cell viability, a dye exclusion test using vital stains like Trypan Blue can be used. Trypan Blue selectively enters dead cells with compromised membranes, staining them blue. Live cells with intact membranes exclude the dye, appearing clear. This differentiates viable from non-viable cells during counting.
Loading the Hemocytometer
Loading the hemocytometer requires careful technique for accurate results. Ensure both the hemocytometer and its specialized coverslip are clean and dry, as dust or moisture interferes with proper loading. The coverslip is thicker than standard microscope coverslips to withstand the surface tension of the liquid and maintain the precise chamber depth.
Place the coverslip over the counting surfaces before applying the sample. Using a micropipette, gently apply a small volume of the well-mixed cell suspension, typically 10 µL, to the edge of the coverslip at the chamber entry notch. Capillary action will draw the suspension evenly into the chamber.
Avoid issues like air bubbles, overfilling, or underfilling. Overfilling can alter the known volume by causing the sample to run into grooves or flood the other chamber. Underfilling results in an incomplete sample. If bubbles occur, clean and dry the hemocytometer thoroughly, then reload the sample.
The Cell Counting Procedure
After loading, place the hemocytometer on a microscope stage and allow cells to settle for a few minutes. Focus on the grid lines using a low power objective, such as 10x magnification, then switch to a higher magnification to visualize individual cells.
A common approach involves counting cells in the four corner large squares and the central large square of the grid. This method provides a representative sample. For very dense suspensions, counting fewer squares or further dilution may be needed.
To avoid double-counting or missing cells, apply consistent rules for cells touching boundary lines. A widely accepted rule is to count cells touching the top and left boundary lines of a square, but exclude those touching the bottom and right lines. This systematic approach minimizes human error and ensures consistency. When using vital stains like Trypan Blue, distinguish between viable (clear) and non-viable (blue) cells, tallying them separately. Aim to count at least 100 cells for statistical significance.
Calculating Cell Concentration
After counting, calculate the cell concentration of the original sample. The formula for cell concentration (cells/mL) uses the average number of cells counted, the dilution factor, and the counting area’s volume.
The standard formula is: Cells/mL = (Average number of cells per square) × (Dilution Factor) × 10,000 cells/mL. The factor 10,000 (or 10^4) is a conversion factor derived from the chamber volume: each large square in an Improved Neubauer hemocytometer has a volume of 0.1 mm^3, and 1 mL equals 1000 mm^3, so 0.1 mm^3 converts to 1/10,000 mL.
For example, if you counted an average of 50 cells per large square and your sample was diluted 1:2 (dilution factor of 2), the calculation is: 50 cells × 2 × 10,000 cells/mL = 1,000,000 cells/mL. This final value represents the cell concentration in the original, undiluted sample. If viability was assessed, calculate live and dead cells separately, then determine viability percentage by dividing viable cells by the total.